Generation and use of polyclonal and monoclonal antibodies specific for 3-phosphohistidine

Abstract

Isolated monoclonal antibodies and or antigen binding fragments thereof are disclosed herein that specifically bind polypeptides comprising a histidine phosphorylated at N3 (3-pHis). Nucleic acids encoding these antibodies, vectors including these nucleic acids, and host cells transformed with these vectors and nucleic acids are also disclosed. Methods are also disclosed for using these antibodies, such as for detection of polypeptides comprising a histidine phosphorylated at N3 (3-pHis). In some embodiments, the methods can be used to investigate signal transduction pathways.

We claim: 
     
       1. An isolated monoclonal antibody or antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region (H-CDR)I, a H-CDR2, and a H-CDR3, and wherein the light chain variable region comprises a light chain complementarity determining region (L-CDR) 1, a L-CDR2, and a L-CDR3, wherein the isolated monoclonal antibody or antigen binding fragment comprises:
 a) the H-CDR1, H-CDR2, and H-CDR3 of the amino acid sequence set forth as SEQ ID NO: 1, and the L-CDR1, L-CDR2, and L-CDR3 of the amino acid sequence set forth as SEQ ID NO: 5; 
 b) the H-CDR1, H-CDR2, and H-CDR3 of the amino acid sequence set forth as SEQ ID NO: 2 and the L-CDR1, L-CDR2, and L-CDR3 of the amino acid sequence set forth as SEQ ID NO: 6; 
 c) the H-CDR1, H-CDR2, and H-CDR3 of the amino acid sequence set forth as SEQ ID NO: 3 and the L-CDR1, L-CDR2, and L-CDR2 of the amino acid sequence set forth as SEQ ID NO: 7; or 
 d) the H-CDR1, H-CDR2, and H-CDR3 of the amino acid sequence set forth as SEQ ID NO: 4, and the L-CDR1, L-CDR2, and L-CDR3 of the amino acid sequence set forth as SEQ ID NO: 8 
 
       wherein the monoclonal antibody specifically binds a polypeptide comprising a histidine phosphorylated at N3 (3-pHis). 
     
     
       2. The isolated monoclonal antibody or antigen binding fragment of  claim 1 , wherein:
 a) the H-CDR1, H-CDR2, and H-CDR3 comprise amino acids 21-28, 45-52, and 88-97 of the amino acid sequence set forth as SEQ ID NO: 1, respectively, and the L-CDR1, L-CDR2, and L-CDR3 comprise amino acids 28-33, 51-53, and 90-102 of the amino acid sequence set forth as SEQ ID NO: 5 respectively; 
 b) the H-CDR1, H-CDR2, and H-CDR3 comprise amino acids 21-28, 46-52, and 91-101 of the amino acid sequence set forth as SEQ ID NO: 2, respectively, and the L-CDR1, L-CDR2, and L-CDR3 comprise amino acids 27-34, 52-54, 91-103 of the amino acid sequence set forth as SEQ ID NO: 6, respectively; 
 c) the H-CDR1, H-CDR2, and H-CDR3 including amino acids 24-31, 49-55, 94-104 of the amino acid sequence set forth as SEQ ID NO: 3, respectively, and the L-CDR1, L-CDR2, and L-CDR3 comprise amino acids 27-34, 52-54, and 91-109 of the amino acid sequence set forth as SEQ ID NO: 7, respectively; or 
 d) the H-CDR1, H-CDR2, and H-CDR3 comprise amino acids 24-31, 49-55, 94-104 of the amino acid sequence set forth as SEQ ID NO: 4, respectively, and the L-CDR1, L-CDR2, and L-CDR3 comprise amino acids amino acids 27-33, 51-53 and 90-102 of the amino acid sequence set forth as SEQ ID NO: 8, respectively. 
 
     
     
       3. The isolated monoclonal antibody or antigen binding fragment of  claim 1 , wherein the heavy chain variable region comprises:
 a) amino acids 1-108 of the amino acid sequence set forth as SEQ ID NO: 1; 
 b) amino acids 1-112 of the amino acid sequence set forth as SEQ ID NO: 2; 
 c) amino acid 1-115 of the amino acid sequence set forth as SEQ ID NO: 3, or 
 d) amino acids 1-115 of the amino acid sequence set forth as SEQ ID NO: 4, respectively. 
 
     
     
       4. The isolated monoclonal antibody or antigen binding fragment of  claim 1 , wherein the light chain variable region comprises:
 a) amino acids 1-113 of the amino acid sequence set forth as SEQ ID NO: 5; 
 b) amino acids 1-114 of the amino acid sequence set forth as SEQ ID NO: 6; 
 c) amino acid 1-120 of the amino acid sequence set forth as SEQ ID NO: 7; or 
 d) amino acids 1-113 of the amino acid sequence set forth as SEQ ID NO: 8, respectively. 
 
     
     
       5. The isolated monoclonal antibody or antigen binding fragment of  claim 1 , wherein:
 a) the heavy chain variable region comprises amino acids 1-108 of the amino acid sequence set forth as SEQ ID NO: 1 and the light chain variable region comprises amino acids 1-113 of the amino acid sequence set forth as SEQ ID NO: 5; 
 b) the heavy chain variable region comprises amino acids 1-112 of the amino acid sequence set forth as SEQ ID NO: 2 and the light chain variable region comprises amino acids 1-114 of the amino acid sequence set forth as SEQ ID NO: 6; 
 c) the heavy chain variable region comprises amino acid 1-115 of the amino acid sequence set forth as SEQ ID NO: 3 and the light chain variable region comprises amino acids 1-120 of the amino acid sequence set forth as SEQ ID NO: 7, or 
 d) the heavy chain variable region comprises amino acids 1-115 of the amino acid sequence set forth as SEQ ID NO: 4 and the light chain variable region comprises amino acids 1-113 of the amino acid sequence set forth as SEQ ID NO: 8. 
 
     
     
       6. The isolated monoclonal antibody or antigen biding fragment of  claim 1 , wherein the monoclonal antibody, or antigen binding fragment, comprises a human framework region. 
     
     
       7. The isolated monoclonal antibody of  claim 1 , wherein the antibody is an IgG. 
     
     
       8. The antigen binding fragment of  claim 1 . 
     
     
       9. The antigen binding fragment of  claim 8 , wherein the antigen binding fragment is a Fv, Fab, F(ab′) 2 , scFV or a scFV 2  fragment. 
     
     
       10. The isolated monoclonal antibody or antigen binding fragment of  claim 1 , conjugated to an effector molecule or a detectable label. 
     
     
       11. The isolated monoclonal antibody or antigen binding fragment of  claim 10 , wherein the detectable marker is a fluorescent, enzymatic, heavy metal or radioactive marker. 
     
     
       12. An isolated nucleic acid molecule encoding the monoclonal antibody or antigen binding fragment of  claim 1 . 
     
     
       13. The isolated nucleic acid molecule of  claim 12  operably linked to a promoter. 
     
     
       14. A vector comprising the nucleic acid molecule of  claim 13 . 
     
     
       15. The vector of  claim 14 , wherein the vector is a plasmid vector or a viral vector. 
     
     
       16. An isolated host cell, comprising the vector of  claim 14 . 
     
     
       17. A composition, comprising an effective amount of the isolated monoclonal antibody or antigen binding fragment of  claim 1 , a nucleic acid molecule encoding the monoclonal antibody or the antigen binding fragment, a vector comprising the nucleic acid molecule, or a host cell expressing the vector, and a pharmaceutically acceptable carrier. 
     
     
       18. A method for detecting the presence of a polypeptide comprising a histidine phosphorylated at N3, comprising:
 contacting a sample comprising polypeptides with an effective amount of the monoclonal antibody or antigen binding fragment of  claim 1  under conditions sufficient to form an immune complex; and 
 detecting the presence of the immune complex, 
 
       wherein the presence of the immune complex indicates the presence of the polypeptide comprising the histidine phosphorylated at N3. 
     
     
       19. The method of  claim 18 , wherein the monoclonal antibody or antigen binding fragment is conjugated to a detectable label. 
     
     
       20. The method of  claim 18 , wherein the polypeptide is present in or obtained from a biological sample from a subject. 
     
     
       21. The method of  claim 18 , wherein the polypeptide comprising the histidine phosphorylated at N3 is a component of a signal transduction pathway. 
     
     
       22. The method of  claim 18 , comprising quantitating the amount of the immune complex.